Enhanced amplification of GC-rich DNA with two organic reagents

Zhang, Zhizhou; Yang, Xia; Li, Meng; Liu, Fang; Shen, Cenchao; Yang, Wenjuan

doi:10.2144/000113203

Cited by 47 publications

(45 citation statements)

References 12 publications

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“…We modified reaction conditions for samples that did not amplify by adding 12 L of polyethylene glycol and 1 L of trehalose to PCRs. The addition of these macromolecules increased reaction efficiency by improving the interaction between reaction components and Taq polymerase (Zimmerman and Harrison 1987;Spiess et al 2004;Zhang et al 2009). Thermal cycling was performed with an initial denaturation at 95°C for 5 min followed by 30 cycles of 94°C for 1 min, 55°C annealing for 1 min, 72°C for 1.5 min, and a final extension of 72°C for 5 min.…”

Section: Mitochondrial Dnamentioning

confidence: 99%

Population genetic structure of the endangered Butler’s Gartersnake (Thamnophis butleri): does the Short-headed Gartersnake (Thamnophis brachystoma) exist in Canada?

et al. 2013

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Understanding population genetic structure is fundamental to conservation of endangered species. It is particularly important when working with species that are morphologically conserved because strong genetic divisions could represent cryptic species. Butler's Gartersnake (Thamnophis butleri (Cope, 1889)) is an endangered species in Canada, having a fragmented distribution and being restricted to southwestern Ontario. Furthermore, it is difficult to distinguish morphologically from a closely related species, the Short-headed Gartersnake (Thamnophis brachystoma (Cope, 1892)). We use mitochondrial DNA (mtDNA) and seven microsatellite DNA loci to evaluate the genetic structure of Canadian T. butleri populations and to test for the presence of T. brachystoma in one of these populations. All individuals had the same mtDNA haplotype, and there was no evidence of multiple, syntopic genetic clusters, thereby rejecting the hypothesis that T. butleri and T. brachystoma co-exist in Canada. Two different model-based assignment tests using microsatellite DNA data suggest that there are four to five genetically distinct clusters of T. butleri (F ST from 0.12 to 0.20). We provide the first population genetic study of T. butleri in Canada and refute the presence of T. brachystoma. Our results may provide guidance on recovery strategies for this species and identify areas to target fine-scale genetic analyses.Résumé : La compréhension de la structure génétique des populations est un aspect fondamental de la conservation des espèces en voie de disparition et est particulièrement importante dans le cas d'espèces morphologiquement conservées étant donné que de fortes divisions génétiques peuvent représenter des espèces cryptiques. La couleuvre à petite tête (Thamnophis butleri (Cope, 1889)) est une espèce en voie de disparition au Canada, son répartition, limitée au sud-ouest de l'Ontario, étant fragmentée. Il est en outre difficile de la distinguer, sur une base morphologique, de Thamnophis brachystoma (Cope, 1892), une espèce étroitement apparentée. Nous utilisons l'ADN mitochondrial (ADNmt) et sept microsatellites d'ADN pour évaluer la structure génétique des populations canadiennes de T. butleri et vérifier la présence de T. brachystoma dans une de ces populations. Tous les individus présentent le même haplotype d'ADNmt, et rien n'indique la présence de multiples groupements génétiques syntopiques, ce qui invalide l'hypothèse de la coexistence de T. butleri et T. brachystoma au Canada. Deux tests d'affectation différents reposant sur des modèles et faisant appel aux données de microsatellites d'ADN laissent croire à la présence de quatre à cinq groupements génétiques distincts de T. butleri (F ST de 0,12 à 0,20). Nous présentons la première étude de la génétique des populations de la couleuvre à petite tête au Canada et démontrons l'absence de la couleuvre T. brachystoma. Nos résultats pourraient servir à orienter les stratégies visant le rétablissement de T. butleri et à cerner les zones à cibler des analyses génétiques fines....

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Section: Mitochondrial Dnamentioning

confidence: 99%

Population genetic structure of the endangered Butler’s Gartersnake (Thamnophis butleri): does the Short-headed Gartersnake (Thamnophis brachystoma) exist in Canada?

et al. 2013

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“…The efficiency of PCR amplification has been reported to be increased by adding organic solvents (17). DMSO and other additives such as betaine and BSA not only reduce the formation of secondary structures like hairpins caused by GC-rich regions but also remove inhibitors present either in the DNA template preparation or in the reaction buffer and increase enzyme stability as well (18)(19)(20)(21)(22). The addition of either DMSO or betaine with BSA in the reaction mixture showed substantial improvement in PCR amplification yield although non-specific amplification products were observed.…”

Section: Discussionmentioning

confidence: 99%

Comparison of the Lipophosphoglycan 3 Gene of the Lizard and Mammalian Leishmania: A Homology Modeling

Pirdel

2016

Res Mol Med (RMM)

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Background: Lipophosphoglycan 3 (LPG3) is required for the LPG assembly, a well known virulent molecule. In this study, the LPG3 gene of the lizard and mammalian Leishmania species were cloned and sequenced. A three-dimensional structure (3D) for the target sequence was also predicted by comparative (homology) modeling. Materials and Methods: An optimization PCR amplification was performed on genomic DNA extracted from two species of Leishmania. The desired PCR products were then cloned and sequenced. In addition, a homology modeling was carried out in order to create three-dimensional structure of the Leishmania LPG3 using SWISS-MODEL server. Results: The GC-rich LPG3 gene of two species of Leishmania was successfully amplified using optimized PCR reaction consisting of betaine and 2-mercaptoethanol with bovine serum albumin and then cloned. Sequence alignment of LPG3 showed 95% identity between Leishmania infantum and lizard Leishmania. With regard to the three-dimensional structure prediction of the modeled sequence of Leishmania LPG3, the similarity was found in the molecular structure. Comparative analysis of functional motifs in the target sequence indicated three conserved domains and a putative C-terminal ER retention signal, as well as several post-translational modification sites. From phylogram, L. infantum was also found to be clustered with lizard Leishmania in the phylogenetic tree. Conclusion: It is suggested that the lizard Leishmania may be evolutionarily more close to L. infantum.

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“…The following five GC-rich targets were investigated: ACE (60% GC-rich), BRAF (74%), GNAQ (79%), GNAS (84%) (Frey, Bachmann et al, 2008) and B4GN4 (78%) (Zhang et al, 2009). PCR reactions were set up in a 50 μL total volume and consisted of 1x PCR buffer (20 mM Tris pH 8.4, 50 mM KCl; Invitrogen), 1.5 mM MgCl 2 (Invitrogen), 2.0 U Taq DNA polymerase (Invitrogen), 0.2 µM primers (TriLink BioTechnologies), 0.2 mM d(A,C,T), X % dGTP, and Y % 7-deaza-dGTP (TriLink Biotechnologies) where X and Y are varying percentages of 0.2 mM (X/Y = 30/70, 25/75, 20/80, 10/90, 0/100).…”

Section: Pcrmentioning

confidence: 99%

Hot Start 7-Deaza-dGTP Improves Sanger Dideoxy Sequencing Data of GC-Rich Targets

Shore¹,

Ashrafi²,

Paul³

2012

DNA Sequencing - Methods and Applications

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Enhanced amplification of GC-rich DNA with two organic reagents

Cited by 47 publications

References 12 publications

Population genetic structure of the endangered Butler’s Gartersnake (Thamnophis butleri): does the Short-headed Gartersnake (Thamnophis brachystoma) exist in Canada?

Population genetic structure of the endangered Butler’s Gartersnake (Thamnophis butleri): does the Short-headed Gartersnake (Thamnophis brachystoma) exist in Canada?

Comparison of the Lipophosphoglycan 3 Gene of the Lizard and Mammalian Leishmania: A Homology Modeling

Hot Start 7-Deaza-dGTP Improves Sanger Dideoxy Sequencing Data of GC-Rich Targets

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